Blood leukocytes migrate towards the sites of inflammation (a beneficial effect) or towards compromised tissues after an ischemic attack (a deleterious effect), such as in the case of ischemia/reperfusion injury (I/RI) in the heart. In I/R, when the supply of blood is restored to the affected area, neutrophils cause damage to healthy tissue via the massive release of reactive oxygen species (ROS). We have discovered that the enzyme Phospholipase D2 (PLD2) is a chemoattractant for neutrophils. In addition to this, we present three major lines of evidence that have allowed us to propose three Specific Aims. (1) First, novel data in our lab has revealed an unexpected GEF activity that exists in the lipase PLD2. Since the target, Rac2 Rho GTPase is responsible for cell movement PLD2 might be a major GEF mediating chemotaxis. Because GEFs are not constitutively active but are kept under tight regulation, we propose in AIM 1 to investigate the regulation of PLD2 novel GEF activity. The hypothesis is that PLD2 GEF activity is regulated by tyrosine phosphorylation and by interaction with PDZ domain proteins, which lead to PX and PH domains of PLD2 and phosphatidic acid (PA) combined action to ensure a GTP/GDP exchange activity. The candidate kinases are VEGFR2 and JAK3, while the PDZ-Domain proteins are MUPP1 and Mda9/Syntenin. (2) Second, we present preliminary evidence of PLD2 as the center of a protein network during cell signaling. We propose in AIM 2 to study a new pathway through which PLD2 and 14-3-3 interact to prevent chemotaxis in ischemia reperfusion injury conditions via GDF-15. We will study the molecular interactions involving PLD2, Rac2, WASp, and the adaptors Grb2 and 14-3-3 through biochemical, genetic (Rac2-/-, WASp-/- and PLD2-/- KO mice) and fluorescence microscopy experiments. Through visualization of protein complex formation in real time by FRET, we will quantify the spatial organization of these signaling molecules during chemotaxis and in I/RI-like conditions. (3) Third, PLD inhibitors protect the heart from injury caused by ischemia/reperfusion in a Murine myocardial/reperfusion injury model, placing PLD at the center of this disease. In AIM 3 we hypothesize that PLD from leukocytes that infiltrate an ischemia/infarct area will have a deleterious effect on the heart conducive to the exacerbation of the injury in vivo. We will test this hypothesis in wild type and PLD2- /- KO mice, as well as in osmotic pump-implanted mice with specific PLD inhibitors. We will also ascertain the PLD regulatory mechanisms of signal transduction that operate in vivo. We expect to demonstrate that depriving PLD confers myocardial protection, with therapeutic potential in conjunction with currently used thrombolytic therapy. This grant will uncover targets that can be exploited pharmacologically to diminish the harmful presence of neutrophils in inflammation-mediated heart injury. If we can avoid the untimely presence of these cells in ischemia, heart failure after myocardial infarction would then be diminished.